S277: IPSC DISEASE MODELS TO STUDY AND TREAT DIAMOND-BLACKFAN ANEMIA SYNDROME (DBAS)
Authors: Chantal Clark, Marije Bartels, Ester Varga, Marieke von Lindern, Emile van den Akker
Journal: HemaSphere (2023)

Background: Diamond-Blackfan Anemia Syndrome (DBAS) is characterized by hypoplastic anemia, congenital malformations and an increased cancer risk. In most patients, DBAS is caused by haploinsufficiency for a ribosomal protein gene, inducing tissue-specific functional defects, including severe anemia. Treatment options are very limited, including glucocorticoids (GC), and blood transfusions, associated with severe toxicity. In a small number of patients, allogeneic stem cell transplantation (SCT) is potentially curative, but not without risks. In addition to the evident need for novel therapeutic options, disease mechanisms driving anemia in DBAS are not completely elucidated, and research is hampered by the scarce availability of patient samples and disease models. Recently, we have developed an induced pluripotent stem cell (iPSC) culture to RBC differentiation platform, validated by necessary functional assays, providing a powerful tool to study all aspects of erythroid development. In addition, this system has been developed to allow scalability and bioreactor RBC production for clinical applications. Aims: As part of the TRACER consortium, we aim to generate novel DBAS models, that can be used to study disease biology, potential novel drugs, and to develop genetically corrected iPSC derived cultured RBCs for transfusion, or iPSC-derived corrected HSCs for autologous SCT (Fig.1A). Methods: Patients were selected from the Dutch Registry (DBAN) based on clinical or molecular characteristics (Fig.1B). The potential of patient peripheral blood mononuclear cell (PBMC) derived CD34+ cells to give rise to proerythroblasts (proEB) in vitro was investigated. Our culture protocol resembles in vivo stress erythropoiesis, using SCF, IL3, GC and EPO during the expansion phase. Cultured proEB were reprogrammed using the non-integrative Cytotune Sendai IPS 2.0 kit that delivers the “Yamanaka factors” footprint-free. The absence of transgene vectors in propagated iPSC clones and the expression of pluripotency markers (e.g. SSEA4, TRA-1-60) was confirmed by RT-PCR and/or flow cytometry. DBAS-iPSC erythroid production is (currently) assessed using our novel differentiation platform. Results: The proEB production potential corresponded to the clinical GC response in two siblings (P1, P2) with GC-refractory DBAS. Their parents (versus healthy controls) also displayed reduced proEB production potential, suggesting asymptomatic carrier status, or other familial factors driving the phenotype in P1 and P2. Cultured proEB from P1, P2 and P5 (RPS26 variant), were successfully reprogrammed to iPSC. In contrast, a GC-unresponsive patient (P4) that showed defective in vitro proEB production potential (mimicking the severe phenotype), also failed to undergo successful reprogramming of PBMCs. Whether reprogramming efficiency of DBAS cells correlates with clinical severity remains to be determined. The capacity of DBAS iPSC clones to produce hematopoietic stem and progenitor cells (HSPC) is currently assessed using our differentiation platform. Whereas DBAS iPSC will most likely display a reduced potential during differentiation (representing disease phenotype), our differentiation platform will likely provide sufficient progenitor cells for further mechanistic studies. Summary/Conclusion: In DBAS there is a clinical need for novel therapeutic strategies to treat severe anemia and reduce organ toxicity. Our previous studies and preliminary data demonstrate that we can produce iPSC lines that provide a sustainable source of DBAS models, in which the regulation of erythropoiesis can be studied in detail, and novel therapeutic strategies, including gene therapy approaches can be tested for clinical applications.Keywords: Diamond-Blackfan anemia, Erythropoieisis, Transfusion, Gene therapy